title: Antiplatelet effects of caffeic acid due to Ca(2＋) mobilizationinhibition via cAMP-dependent inositol-1, 4, 5-trisphosphate receptor phosphorylation.
abstract: OBJECTIVE
  In this study, we investigated the effects of caffeic acid (CAFA), a phenolic acid, on Ca(2＋)-antagonistic cyclic nucleotides associated with the phosphorylation of inositol 1,4,5-trisphosphate receptor (IP3R) and vasodilator-stimulated phosphoprotein (VASP) and the thromboxane A2 (TXA2)-associated microsomal cyclooxygenase-1 (COX-1) activity in collagen (10 μg/mL)-stimulated platelet aggregation.
  
  
  METHODS
  Washed platelets (10(8)/mL) obtained from Sprague-Dawley rats (6-7 weeks old, male) were preincubated for 3 minutes at 37℃ in the presence of 2 mM exogenous CaCl2 with or without CAFA or other materials, stimulated with collagen (10 μg/mL) for 5 minutes, then used to determine the levels of intracellular cytosolic Ca(2＋) ([Ca(2＋)]i), TXA2, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), COX-1 activity, VASP and IP3R phosphorylation.
  
  
  RESULTS
  CAFA dose-dependently inhibited collagen-induced platelet aggregation and suppressed the production of TXA2, an aggregation-inducing autacoid associated with the strong inhibition of COX-1 in platelet microsomes exhibiting cytochrome C reductase activity. CAFA dose-dependently inhibited collagen-elevated [Ca(2＋)]i mobilization, which was increased by a cAMP-dependent protein kinase (A-kinase) inhibitor, Rp-8-Br-cAMPS, but not a cGMP-dependent protein kinase (G-kinase) inhibitor, Rp-8-Br-cGMPS. In addition, CAFA significantly increased the formation of cAMP and cGMP, intracellular Ca(2＋)-antagonists that function as aggregation-inhibiting molecules. CAFA increased IP3R (320 kDa) phosphorylation, indicating the inhibition of IP3-mediated Ca(2＋) release from internal stores (i.e. the dense tubular system) via the IP3R on collagen-activated platelets. Furthermore, CAFA-induced IP3R phosphorylation was strongly inhibited by an A-kinase inhibitor, Rp-8-Br-cAMPS, but only mildly inhibited by a G-kinase inhibitor, Rp-8-Br-cGMPS. These results suggest that the inhibition of [Ca(2＋)]i mobilization by CAFA is resulted from the cAMP/A-kinase-dependent phosphorylation of IP3R. CAFA elevated the phosphorylation of VASP-Ser(157), an A-kinase substrate, but not the phosphorylation of VASP-Ser(239), a G-kinase substrate. We demonstrate that CAFA increases cAMP and subsequently phosphorylates both IP3R and VASP-Ser(157) through A-kinase activation to inhibit [Ca(2＋)]i mobilization and TXA2 production via the inhibition of the COX-1 activity.
  
  
  CONCLUSIONS
  These results strongly indicate that CAFA is a potent beneficial compound that elevates the level of cAMP-dependent protein phosphorylation in collagen-platelet interactions, which may result in the prevention of platelet aggregation-mediated thrombotic diseases.
authors.affiliation: Department of Biomedical Laboratory Science, College of Biomedical Science and Engineering, Inje University.
authors.forename: Dong-Ha
authors.initials: DH
authors.lastname: Lee
authors.forename: Hyun-Hong
authors.initials: HH
authors.lastname: Kim
authors.forename: Hyun-Jeong
authors.initials: HJ
authors.lastname: Cho
authors.forename: Jeong-Soo
authors.initials: JS
authors.lastname: Bae
authors.forename: Young-Bin
authors.initials: YB
authors.lastname: Yu
authors.forename: Hwa-Jin
authors.initials: HJ
authors.lastname: Park
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D015153:Blotting, Western; D002109:Caffeic Acids / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D051545:Cyclooxygenase 1 / Q000378:metabolism; D004594:Electrophysiology; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013928:Thromboxane A2 / Q000378:metabolism; D000098662:Vasodilator-Stimulated Phosphoprotein
chemical_list: D002109:Caffeic Acids; D015815:Cell Adhesion Molecules; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008565:Membrane Proteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; D010975:Platelet Aggregation Inhibitors; D000098662:Vasodilator-Stimulated Phosphoprotein; D013928:Thromboxane A2; D000242:Cyclic AMP; D051545:Cyclooxygenase 1; C496513:Ptgs1 protein, rat; D002118:Calcium; C040048:caffeic acid